Compositions in which an alkyl or aryl substituent is affixed to the surface of siliceous materials by covalent chemical bonding with the resultant bonded materials being used for chromatography are known to the prior art. Exemplary of this type of prior art is Locke et al., Analytical Chemistry, Vol. 44, No. 1, pages 90-92 (1972). It is also known to react a siliceous surface with monohalogenated silanes, silazines or silylamines, or with monoalkoxy or monoacetoxysilanes, and then to cause a chemical modification of the reaction product. This type of prior art is illustrated by U.S. Pat. No. 4,043,905 to Novotny et al.
The use of tetra-substituted amino silane compositions covalently bonded to the surface of siliceous materials is also known. Majors, Journal of Chromatographic Science, Vol. 12, 767 (1974), used such a bonded phase in the "normal" mode for separation of azo compounds and polar steroids using organic solvents.
Partial resolution of biologically important mixtures of adenosine monophosphate isomers was achieved by Arendes et al., Journal of Chromatography, Vol. 140, 118 (1977).
Separation of the 2'- and 3'-isomers of adenosine monophosphate and guanosine monophosphate was achieved by Kratovich and Roe, Journal of Chromatography, Vol. 155, 407 (1978), using a strong anion exchange resin column.
Complete resolution of 2'-, 3'- and 5'-cytidine monophosphate was achieved in 15 hours by Ponnamperuma and Mack, Science, Vol. 148, 1221 (1965), using a Dowex-1 formate column. Further, Khym Clinical Chemistry, Vol. 21, 1245 (1975); Journal of Chromatography, Vol. 124, 415 (1976), used a gradient elution to achieve separation of ribo- from deoxyribonucleotides; however, complete elution of all nucleotides required 1-3 hours, and some pairs of ribo- and deoxyribonucleoside monophosphates were poorly resolved.
Brown et al., Journal of Chromatography, Vol. 112, 651 (1975); Vol. 152, 253 (1978), achieved separation of ribo- and deoxyribonucleoside mono-, di- and triphosphates using a silica-based, strong anion exchanger, but were not able to simultaneously obtain good resolution and a satisfactory analysis time.
Thus, although the prior art has achieved simultaneous resolution of some members of biologically important classes, it has not been able to rapidly and simultaneously separate bases, nucleosides and nucleotides, and isomers thereof, present in biological samples.